Gene delivery vectors with cell type specificity for primary human chondrocytes

ABSTRACT

The present invention relates to a gene delivery vehicle comprising a recombinant adenovirus having a tropism for a primary human chondrocyte. By efficiently transducing a nucleic acid of interest into a primary chondrocytes, the gene delivery vehicle is able, at least in part, to improve the cartilage disease. In one embodiment, the recombinant adenovirus has a deletion in the gene encoding the fiber protein, which is replaced by a nucleic acid sequence encoding at least part of a fiber protein of a B-type adenovirus.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of application Ser. No.09/928,262, filed Aug. 10, 2001, pending, which claimed priority fromU.S. Provisional Patent ApplicationNo. 60/224,911 filed on Aug. 11,2000, the contents of both of which are incorporated by this reference.

TECHNICAL FIELD

[0002] The invention relates to the field of molecular genetics andmedicine. In particular the present invention relates to the field ofgene therapy, more in particular to gene therapy using adenoviruses.

BACKGROUND

[0003] At present in gene therapy, genetic information is delivered to ahost cell in order to either correct (supplement) a genetic deficiencyin the cell, or to inhibit an unwanted function in the cell, or toeliminate the host cell. Of course, the genetic information can also beintended to provide the host cell with a wanted function, for instanceto supply a secreted protein to treat other cells of the host, etc.Thus, there are at least three different approaches in gene therapy, onedirected towards compensating a deficiency present in a (mammalian)host; the second directed towards the removal or elimination of unwantedsubstances (organisms or cells); and the third towards providing a cellwith a wanted function.

[0004] For the purpose of gene therapy, adenoviruses have been proposedas suitable vehicles to deliver genes to the host. Gene-transfer vectorsderived from adenoviruses (so-called adenoviral vectors) have a numberof features that make them particularly useful for gene transfer. 1) Thebiology of the adenoviruses is characterized in detail, 2) theadenovirus is not associated with severe human pathology, 3) the virusis extremely efficient in introducing its DNA into the host cell, 4) thevirus can infect a wide variety of c ells and has a broad host-range, 5)the virus can be produced at high virus titers in large quantities, and6) the virus can be rendered replication defective by deletion of theearly-region 1 (E1) of the viral genome (Brady and Crystal 1994).

[0005] However, there are still drawbacks associated with the use ofadenoviral vectors especially the well investigated serotypes ofsubgroup C adenoviruses. These serotypes require the presence of theCoxsackie adenovirus receptor (CAR) on cells for successful infection.Although this protein is expressed by many cells and established celllines, this protein is absent on many other primary cells and cell linesmaking the latter cells difficult to infect with serotypes 1, 2, 5, and6.

[0006] The adenovirus genome is a linear double-stranded DNA molecule ofapproximately 36000 base pairs. The adenovirus DNA contains identicalInverted Terminal Repeats (ITRs) of approximately 90-140 base pairs withthe exact length depending on the serotype. The viral origins ofreplication are within the ITRs exactly at the genome ends. Mostadenoviral vectors currently used in gene therapy have a deletion in theE1 region, where novel genetic information can be introduced. The E1deletion renders the recombinant virus replication defective. It hasbeen demonstrated extensively that recombinant adenovirus, in particularserotype 5 is suitable for efficient transfer of genes in vivo to theliver, the airway epithelium and solid tumors in animal models and humanxenografts in immunodeficient mice (Bout 1996; Blaese et al. 1995). Atpresent, six different subgroups of human adenoviruses have beenproposed which in total encompasses 51 distinct adenovirus serotypes.Besides these human adenoviruses an extensive number of animaladenoviruses have been identified (Ishibashi and Yasue 1984).

[0007] A serotype is defined on the basis of its immunologicaldistinctiveness as determined by quantitative neutralization with animalantisera (horse, rabbit). If neutralization shows a certain degree ofcross-reaction between two viruses, distinctiveness of serotype isassumed if A) the hemagglutinins are unrelated, as shown by lack ofcross-reaction on hemagglutination-inhibition, or B) substantialbiophysical/biochemical differences in DNA exist (Francki et al. 1991).The nine serotypes identified last (42-51) were isolated for the firsttime from HIV-infected patients (Hierholzer et al. 1988; Schnurr andDondero 1993; De Jong et al. 1999). For reasons not well understood,most of such immuno-compromised patients shed adenoviruses that wererarely or never isolated from immuno-competent individuals (Hierholzeret al. 1988; Hierholzer 1992; Khoo et al. 1995, De Jong et al. 1999).

[0008] At present, the adenovirus serotype 5 is most widely used forgene therapy purposes. Similar to serotypes 2, 4 and 7, serotype 5 has anatural affiliation towards lung epithelia and other respiratorytissues. In contrast, it is known that, for instance, serotypes 40 and41 have a natural affiliation towards the gastrointestinal tract. For adetailed overview of the disease association of the different adenovirusserotypes see Table 1. In this Table 1 there is one deviation from theliterature. Sequence analysis and hemagglutination assays usingerythrocytes from different species performed in our institute indicatedthat in contrast to the literature (De Jong et al. 1999) adenovirus 50proved to be a D group vector whereas adenovirus 51 proved to be aB-group vector.

[0009] The natural affiliation of a given serotype towards a specificorgan can either be due to a difference in the route of infection, i.e.,make use of different receptor molecules or internalization pathways.However, it can also be due to the fact that a serotype can infect manytissues/organs but it can only replicate in one organ because of therequirement of certain cellular factors for replication and henceclinical disease. At present, it is unknown which of the foregoingmechanisms is responsible for the observed differences in human diseaseassociation. However, it is known that different adenovirus serotypescan bind to different receptors due to sequence dissimilarity of thecapsid proteins, for example, fiber proteins. For instance, it has beenshown that adenoviruses of subgroup C such as Ad2, and Ad5 bind todifferent receptors as compared to adenoviruses from subgroup B such asAd3 (Defer et al. 1990). An adenovirus from subgroup B is referred to asa B-type adenovirus. Likewise, it was demonstrated that receptorspecificity could be altered by exchanging the Ad3 with the Ad 5 knobprotein, and vice versa (Krasnykh et al. 1996; Stevenson et al. 1995 and1997). The C-terminus of the fiber protein, or knob, is responsible forinitial interaction with the cellular adenovirus receptor. Thus, thefiber protein is mainly responsible for receptor specificity. Asdifferent host cells can have different receptors, the fiber proteinlargely determines at which host cells the adenovirus preferably binds.The preference for binding to a certain kind of host cell is called atropism. If an adenovirus has a tropism for a certain host cell, it may,or may not, bind to other kind of cells as well. The tropism of anadenovirus is thus at 1 east partly dependent on the kind of fiberprotein, and/or knob protein.

[0010] In the United States alone, 95,000 knee replacements and 41,000other surgical procedures to repair cartilaginous defects of the kneeare performed on an annual basis. This, together with other cartilagediseases (i.e., joint surface irregularities, craniofacial deformation,osteogenesis imperfecta, meniscal injury, anencephaly, intra articularfractures, osteoporosis, osteoarthritis, spinal cord fusion, andrheumatoid arthritis) warrant the enormous interest in understanding theunderlying biological and biochemical defects of the diseases as well asthe interest in gene therapy as a possible cure (reviewed in Frenkel andDi Cesare 1999). The strategies to treat these diseases are diverseranging from direct delivery of genes to sites of injuries, tocell-based delivery approaches, or ex vivo tissue engineering. In casegenes are directly delivered to a site of injury either retroviruses,adenoviruses, naked DNA, or liposome complexed DNA are contemplated(Madry and Trippel 2000; Lubberts et al. 1999; Goto et al 1999). The DNAcan encode either for amino acid sequences that inhibit the diseaseprogression and/or amino acid sequences that counteract the loss ofcartilage. Non-limiting examples of genes that inhibit diseaseprogression are TGF beta (Nishida et al. 1999), IL-4 (Lubberts et al.1999), p16INK4a (Taniguchi et al. 1999), IL-1 (Fernandez et al. 1999),IL-10 (Whalen et al. 1999), or substances (anti-inflammatory drugs,TNFa, immunosuppressive agents) which can down regulate the activity ofNOS or COX (Amin et al. 1999). Both other strategies—cell based or exvivo bioengineering—use the same genes (Gazit et al. 1999): twopleiotropic inflammatory mediators overproduced in arthritis infectedjoints. Non-limiting examples of genes that counteract the cartilagedegradation are the family of bone morphogenesis proteins (Mason et al.1998; Kramer et al 2000, Pizette and Niswander 2000). Cells of choice toperform cell based delivery at sites of injury or transplanted into ascaffold are chondrocytes or mesenchymal stem cells derived from humanbone marrow (Richardson et al. 1999, Silverman et al. 2000, Gazit et al.1999). For all these strategies to become therapeutically interesting,the delivery of gene sequences chondrocytes needs to be very efficientsuch that a) expression levels of the therapeutic genes are high and b)low dosages of the gene transfer vehicle, i.e., adenoviruses can beapplied to circumvent possible vector-mediated toxic effects.

DISCLSOSURE OF THE INVENTION

[0011] The present invention addresses the problem of how cartilagediseases can be counteracted by efficiently transducing a nucleic acidinto primary chondrocytes. For that purpose, a gene delivery vehiclecomprising a recombinant adenovirus having a tropism for primary humanchondrocytes has been constructed. By a “gene delivery vehicle” is meanta carrier that can deliver at least one nucleic acid to a host cell. Thenucleic acid that is delivered to a host cell may comprise a nucleicacid sequence encoding an amino acid sequence. The nucleic acid mayfurther comprise at least one promoter, and/or enhancer, and/orterminator. It may also comprise transcription initiation sites, and thelike.

[0012] By delivering a nucleic acid to a host cell, the nucleic acid ismoved from the outside to the inside of the host cell. Transientexpression of the transgene is sufficient to trigger cells to form bone,or trigger angiogenesis. Therefore, a non-integrating vector ispreferred, for example, adenovirus. The present invention shows thatprimary human chondrocytes do not express detectable levels of CAR orMHC-class I, as is described in Example 4. The latter indicates that itis difficult to transduce primary human chondrocytes with an adenovirusthat enters the cells via these molecules, as for example thecommonly-used adenovirus serotype 5. One may use very high titers of theadenovirus, but this has several disadvantages such as a strong immuneresponse caused by de novo synthesis of adenoviral genes that cansubsequently by loaded in MHC class I complexes and presented to theimmune system once the cells are transplanted in a host. To avoid toxicside effects, one would like to be able to transfer a nucleic acid toprimary human chondrocytes by a gene delivery vehicle with highefficacy. High efficiency of infection allows for a reduction in theviral load that results in less virus binding to cells other than thetarget cells of interest. If the gene delivery vehicle infects too manyother cells, the expression of the delivered nucleic acid in those othercells may cause many side effects.

[0013] Therefore, the present invention discloses a gene deliveryvehicle which has been made specific for a primary chondrocyte and whichhas the other properties of adenoviruses, for example not integratingits DNA in the host cell genome. To provide specificity forchondrocytes, the present invention discloses an adenovirus thatcomprises a deletion in the gene encoding for fiber protein that isreplaced by a nucleic acid sequence encoding an amino acid sequencehaving a tropism for primary human chondrocytes. The nucleic acidsequence encoding an amino acid sequence having a tropism for primaryhuman chondrocytes may be derived from any gene encoding for fiberprotein. It may comprise at least one mutation that makes it differentfrom any wild type gene encoding for fiber protein. Otherwise, thenucleic acid may be an unmodified gene encoding for fiber protein of anyserotype. If the adenovirus disclosed herein includes nucleic acidsequences of at least two different serotypes, the adenovirus isreferred to as a “chimeric adenovirus”.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0014]FIG. 1: Expression of CAR, MHC-class I, and a_(v)-integrins onprimary chondrocytes. As a control for the antibodies, PER.C6 cells weretaken along.

[0015]FIG. 2: Screening the fiber chimeric viruses for the presence ofviruses that are better suited for transduction of primary humanchondrocytes. The doses used are 100, 500 or 1000 virus particles percell. Luciferase activity is expressed in relative light units (RLU).(A) and (B) represent two separate independent experiments performed onchondrocytes derived from different donors.

[0016]FIG. 3: (A) Effect of adenoviral infection on chondrocyteviability. Three different moi's of a LacZ expressing virus(Ad5Fib16LaCZ, generated with pCIJP.LacZ as adapter plasmid) were usedto infect human primary chondrocytes. Viability was checked at severaltime points following the infection. (B) Duration of LacZ expression intransfected human chondrocytes.

[0017]FIG. 4: (A) Flow cytometric analysis of chondrocytes exposed todifferent adenoviral vectors. Non-transduced chondrocytes were used toset the flow cytometric background at 1% Green Fluorescent Protein (GFP)positive cells. Shown is the percentage of chondrocytes in the cellpopulation that became positive for GFP after infection with GFPexpressing recombinant adenoviruses (X in AdFibXAdaptGFP stands fordifferent fibers derived from different serotypes in a Ad5 backbonegenerated with pAdApt as adapter plasmid). (B) Shown is the medianfluorescence that indicates the amount of GFP produced per cell.

DETAILED DESCRIPTION OF THE INVENTION

[0018] Although primary human chondrocytes do not express detectablelevels of CAR protein, the adenovirus disclosed in the present inventionis well capable of infecting the chondrocytes. Therefore it is possibleto use a recombinant, adenovirus that is derived from an adenovirusserotype 5 sequence, although an adenovirus serotype 5 normally does notinfect primary chondrocytes. The recombinant adenovirus may comprise anadenovirus 5 nucleic acid sequence. It may comprise an adenovirus 5genome, comprising at least one deletion in its E1 region where anucleic acid of interest is inserted or can be inserted.

[0019] In the counteraction of cartilage diseases, the nucleic acidwhich is delivered to primary human chondrocytes preferably eitherencodes an amino acid sequence that inhibits cartilage diseaseprogression or a amino acid sequence that counteracts the loss ofcartilage. The nucleic acid can encode a member of the family of bonemorphogenesis proteins. Alternatively, the nucleic acid can encode anamino acid sequence which provides the host cell with another wantedfunction.

[0020] Another feature of the present invention is the means to producea chimeric virus. Typically, one does not want an adenovirus batch to beadministered to a host cell that contains replication competentadenovirus, although this is not always true. In general therefore it isdesired to omit a number of genes (but at least one) from the adenoviralgenome on the vector encoding the chimeric virus and to supply thesegenes in the genome of the cell in which the vector is brought toproduce chimeric adenovirus. Such a cell is usually called a packagingcell. The invention thus also provides a packaging cell for producing achimeric adenovirus according to the invention, comprising in trans allelements necessary for adenovirus production not present on theadenoviral vector according to the invention. Typically vector andpackaging cell have to be adapted to one another in that they have allthe necessary elements, but that they do not have overlapping elementswhich lead to replication competent virus by recombination.

[0021] The initial step for successful infection is binding ofadenovirus to its target cell, a process mediated through fiber protein.The fiber protein has a trimeric structure (Stouten et al. 1992) withdifferent lengths depending on the virus serotype (Signas et al. 1985;Kidd et al. 1993). Different serotypes have polypeptides withstructurally similar N and C termini, but different middle stem regions.N-terminally, the first 30 amino acids are involved in anchoring of thefiber to the penton base (Chrobcczek et al. 1995), especially theconserved FNPVYP (SEQ ID NO: 14)region in the tail (Arnberg et al.1997). The knob is responsible for initial interaction with the cellularadenovirus receptor. After this initial binding secondary bindingbetween the capsid penton base and cell-surface integrins is proposed tolead to internalization of viral particles in coated pits andendocytosis (Morgan et al. 1969; Svensson and Persson 1984; Varga et al.1991; Greber et al. 1993; Wickham et al, 1993).

[0022] Integrins are αβ-heterodimers of which at least 14 αsubunits and8 β-subunits have been identified (Hynes 1992). The array of integrinsexpressed in cells is complex and will vary between cell types andcellular environment. Although the knob contains some conserved regions,between serotypes, knob proteins show a high degree of variability,indicating that different adenovirus receptors might exist. Forinstance, it has been demonstrated that adenoviruses of subgroup C (Ad2,Ad5) and adenoviruses of subgroup B (Ad3) bind to different receptors(Defer et al. 1990). By using baculovirus produced soluble CAR as wellas adenovirus serotype 5 knob protein, Roelvink et al. (1998) concludedvia interference studies that all adenovirus serotypes, except serotypesof subgroup B, enter cells via CAR. The latter, if valid, limits thecomplexity of using different serotypes for gene therapy purposes.

[0023] Besides the involvement in cell binding, the fiber protein alsocontains the type specific γ-antigen, which together with the ε-antigenof the hexon determines the serotype specificity. The γ-antigen islocalized on the fiber and it is known that it consists of 17 aminoacids. The anti-fiber antibodies of the host are therefore directed tothe trimeric structure of the knob.

[0024] The present invention provides a method and means by which anadenovirus can infect primary human chondrocytes. Therefore, thegeneration of preferably chimeric adenoviruses based for example onadenovirus serotype 5 with modified fiber genes, is described. For thispurpose, two or three plasmids, which together contain the completeadenovirus serotype 5 genome, were constructed. From this plasmid, theDNA encoding the adenovirus serotype 5 fiber protein was removed andreplaced by linker DNA sequences which facilitate easy cloning. Theplasmid in which the native adenovirus serotype 5 fiber sequence waspartially removed subsequently served as a template for the insertion ofDNA encoding for fiber protein derived from different adenovirusserotypes (human or animal). The DNAs derived from the differentserotypes were obtained using the polymerase chain reaction technique incombination with (degenerate) oligonucleotides. At the former E1location in the genome of adenovirus serotype 5, any nucleic acid ofinterest can be cloned. A single transfection procedure of the two orthree plasmids together resulted in the formation of a recombinantchimeric adenovirus. Although successful introduction of changes in theadenovirus serotype 5 fiber and penton-base have been reported byothers, the complex structure of knob and the limited knowledge of theprecise amino acids interacting with CAR render such targetingapproaches laborious and difficult.

[0025] To overcome the limitations described above, we preferred to usepre-existing adenovirus fibers to maximize the chance of obtainingrecombinant adenovirus which can normally assemble in the nucleus of aproducer cell and which can be produced on pre-existing packaging cells.By generating for example a chimeric adenovirus serotype 5 based fiberlibrary containing fiber proteins of all other human adenovirusserotypes, we have developed a technology which enables rapid screeningfor a recombinant adenoviral vector with preferred infectioncharacteristics for primary human chondrocytes.

[0026] In another aspect the invention describes the construction anduse of plasmids consisting of distinct parts of for example adenovirusserotype 5 in which the gene encoding for fiber protein has beenreplaced with DNA derived from alternative human or animal serotypes.This set of constructs, in total encompassing the complete adenovirusgenome, allows for the construction of unique chimeric adenovirusescustomized for transduction of particular cell types or organ(s). Also,in this part of the invention means and methods to propagate, produce,and purify fiber chimeric adenoviruses are described.

[0027] In another aspect of the invention, chimeric viruses aredescribed which have preferred infection characteristics in humanprimary chondrocytes. The adenoviral vectors preferably are derived fromsubgroup B adenoviruses or contain at least a functional part of thefiber protein from an adenovirus from subgroup B comprising at least thebinding moiety of the fiber protein. In a further preferred embodiment,the adenoviral vectors are chimeric vectors based on adenovirus serotype5 and contain at least a functional part of the fiber protein fromadenovirus type 16, 35, or

[0028] 51. Although adenovirus serotype 5 does not bind to primarychondrocytes, the binding moiety of an adenoviral fiber protein of aB-type adenovirus appears to be sufficient to make the chimericadenovirus efficiently infect primary chondrocytes. It is to beunderstood that in all embodiments the adenoviral vectors may be derivedfrom the serotype having the desired properties or that the adenoviralvector is based on an adenovirus from one serotype and contains thesequences comprising the desired functions of another serotype, thesesequences replacing the native sequences in the first serotype.

[0029] In another aspect of the invention, the recombinant adenovirusesmay, or may not, contain deletions in the E1 region where a nucleic acidof interest is inserted or can be inserted. Furthermore, chimericadenoviruses may, or may not, contain deletions in the E3, E2 and/or E4region where a nucleic acid of interest is inserted or can be inserted.If the nucleic acid of interest does not comprise a promoter, it shouldbe linked to a promoter if the nucleic acid of interest is inserted inthe E3, E2 and/or E4 region. If the recombinant adenovirus comprisesdeletions in the E2 and/or E4 region, E2 and/or E4 complementing celllines are required to generate recombinant adenoviruses.

[0030] The present invention also provides a gene delivery vehiclehaving a tropism for primary human chondrocytes comprising a recombinantadenovirus. This recombinant adenovirus may be a chimeric adenovirus.The recombinant adenovirus may contain a deletion in the gene encodingfor fiber protein that is replaced by a nucleic acid encoding an aminoacid sequence having a tropism for primary human chondrocytes. As isdescribed herein, the tropism may be provided by at least a tropismdetermining part of an adenoviral fiber protein of a B-type adenovirus,and the fiber protein may be derived from an adenovirus type 16, 35and/or 51. The recombinant adenovirus may comprise an adenovirus 5nucleic acid sequence and it may comprise an adenovirus 5 genome whichat least has a deletion in its E1 region where a nucleic acid ofinterest is inserted or can be inserted. And it may comprise deletionsin its E3, E2 and/or E4 region, where a nucleic acid of interest isinserted or can be inserted, as is described in this application. Thisrecombinant adenovirus may comprise a nucleic acid sequence encoding atleast one amino acid sequence that inhibits cartilage diseaseprogression and/or at least one amino acid sequence that counteracts theloss of cartilage. It may comprise a nucleic acid that encodes at leastone member of the family of bone morphogenesis proteins. Also, thenucleic acid may provide the host cell with another wanted function.

[0031] The present invention also provides a pharmaceutical compositionfor use in treatment of cartilage diseases. This pharmaceuticalcomposition comprises a gene delivery vehicle as described in thepreceding paragraph. An advantage of this pharmaceutical composition isthat it can counteract cartilage diseases in a very efficient way,because it comprises a gene delivery vehicle that infects chondrocytesvery efficiently. Because of this efficiency, a small amount will besufficient. Therefore, there should be few side effects. The immuneresponse will be low. Besides, the pharmaceutical composition comprisesa non-integrating vector, which is to be preferred to avoid genometransformation of the host cell and its offspring. Because thispharmaceutical composition comprises a well-known non-integratingvector, it is possible to produce this composition, with the learning ofthis invention, on a large scale.

EXAMPLES

[0032] The following examples are meant to illustrate the presentinvention. They are not limiting the present invention. A person skilledin the art can perform alternative experiments that are still in thescope of the present invention. The generation of adenovirus serotype 5genomic plasmid clones and adenovirus serotype 5 based viruses withchimeric fiber proteins are described. Then, primary chondrocytes aretested for expression of integrins and CAR protein. Finally,transduction of human primary chondrocytes with recombinant fiberchimeric adenoviruses is determined.

Example 1 Generation of Adenovirus Serotype 5 Genomic 15 Plasmid Clones

[0033] The complete genome of adenovirus serotype 5 has been cloned intovarious plasmids or cosmids to allow easy modification of parts of theadenovirus serotype 5 genome, still retaining the capability to producerecombinant virus. For this purpose the following plasmids weregenerated:

[0034] 1. pBr/Ad.Bam-rITR (ECACC deposit P97082122)

[0035] In order to facilitate blunt end cloning of the ITR sequences,wild-type human adenovirus type 5 (Ad5) DNA was treated with Klenowenzyme in the presence of excess dNTPs. After inactivation of the Klenowenzyme and purification by phenol/chloroform extraction followed byethanol precipitation, the DNA was digested with BamHI. This DNApreparation was used without further purification in a ligation reactionwith pBr322 derived vector DNA prepared as follows: pBr322 DNA wasdigested with EcoRV and BamHI, dephosphorylated by treatment with TSAPenzyme (Life Technologies) and purified on LMP agarose gel (Sea PlaqueGTG). After transformation into competent E. coli DH5α (Life Techn.) andanalysis of ampicillin resistant colonies, one clone was selected thatshowed a digestion pattern as expected for an insert extending from theBamHI site in Ad5 to the right ITR. Sequence analysis of the cloningborder at the right ITR revealed that the most 3′ G residue of the ITRwas missing, the remainder of the ITR was found to be correct. Themissing G residue is complemented by the other ITR during replication.

[0036] 2. pBr/Ad.Sal-rITR (ECACC deposit P97082119)

[0037] pBr/Ad.Bam-rITR was digested with BamHI and SalI. The vectorfragment including the adenovirus insert was isolated in LMP agarose(Sea Plaque GTG) and ligated to a 4.8 kb SalI-BamHI fragment obtainedfrom wt Ad5 DNA and purified with the Geneclean II kit (Bio 101, Inc).One clone was chosen and the integrity of the Ad5 sequences wasdetermined by restriction enzyme analysis. Clone pBr/Ad.Sal-rITRcontains adeno type 5 sequences from the SalI site at bp 16746 up to andincluding the rITR (missing the most 3′ G residue).

[0038] 3. pBr/Ad.Cla-Bam (ECACC deposit P97082117)

[0039] Wild type Adenovirus type 5 DNA was digested with ClaI and BamHI,and the 20.6 kb fragment was isolated from gel by electro-elution.pBr322 was digested with the same enzymes and purified from agarose gelby Geneclean. Both fragments were ligated and transformed into competentDH5α. The resulting clone pBr/Ad.Cla-Bam was analyzed by restrictionenzyme digestion and shown to contain an insert with adenovirussequences from bp 919 to 21566.

[0040] 4. pBr/Ad.AflII-Bam (ECACC deposit P97082114)

[0041] Clone pBr/Ad.Cla-Bam was linearized with EcoRI (in pBr322) andpartially digested with AflII. After heat inactivation of AflII for 20′at 65° C. the fragment ends were filled in with Klenow enzyme. The DNAwas then ligated to a blunt double stranded oligo linker containing aPacI site (5-AATTGTCTTAATTAACCGCTTAA-3′ (SEQ ID NO:1)). This linker wasmade by annealing the following two oligonucleotides:5′-AATTGTCTTAATTAACCGC-3′ (SEQ ID NO:2) and 5′-AATTGCGGTTAATTAAGAC-3′(SEQ ID NO:3), followed by blunting with Klenow enzyme. Afterprecipitation of the ligated DNA to change buffer, the ligatioris weredigested with an excess PacI enzyme to remove concatameres of the oligo.The 22016 bp partial fragment containing Ad5 sequences from bp 3534 upto 21566 and the vector sequences, was isolated in LMP agarose (SeaPlaque GTG), religated and transformed into competent DH5α. One clonethat was found to contain the PacI site and that had retained the largeadeno fragment was selected and sequenced at the 5′ end to verifycorrect insertion of the PacI linker in the (lost) AflII site.

[0042] 5. pBr/Ad.Bam-rITRpac#2 (ECACC deposit P97082120) andpBr/Ad.Bam-rITR#8 (ECACC deposit P97082121)

[0043] To allow insertion of a PacI site near the ITR of Ad5 in clonepBr/Ad.Bam-rITR, about 190 nucleotides were removed between the ClaIsite in the pBr322 backbone and the start of the ITR sequences. This wasdone as follows: pBr/Ad.Bam-rITR was digested with ClaI and treated withnuclease Bal31 for varying lengths of time (2′, 5′, 10′ and 15′). Theextent of nucleotide removal was followed by separate reactions onpBr322 DNA (also digested at the ClaI site), using identical buffers andconditions. Bal31 enzyme was inactivated by incubation at 75° C. for 10min, the DNA was precipitated and resuspended in a smaller volume of TEbuffer. To ensure blunt ends, DNAs were further treated with T4 DNApolymerase in the presence of excess dNTPs. After digestion of the(control) pBr322 DNA with SalI, satisfactory degradation (˜150 bp) wasobserved in the samples treated for 10 min or 15 min. The 10 min or 15min treated pBr/Ad.Bam-rITR samples were then ligated to the abovedescribed blunted PacI linkers (See pBr/Ad.AflII-Bam). Ligations werepurified by precipitation, digested with excess PacI and separated fromthe linkers on an LMP agarose gel. After religation, DNAs weretransformed into competent DH5a and colonies analyzed. Ten clones wereselected that showed a deletion of approximately the desired length andthese were further analyzed by T-track sequencing (T7 sequencing kit,Pharmacia Biotech). Two clones were found with the PacI linker insertedjust downstream of the rITR. After digestion with PacI, clone #2 has 28bp and clone #8 has 27 bp attached to the ITR.

[0044] pWE/Ad.AflII-rITR (ECACC deposit P97082116)

[0045] Cosmid vector pWE15 (Clontech) was used to clone larger Ad5inserts. First, a linker containing a unique PacI site was inserted inthe EcoRI sites of pWE15 creating pWE.pac. To this end, the doublestranded PacI oligo as described for pBr/Ad.AflII-BamHI was used but nowwith its EcoRI protruding ends. The following fragments were thenisolated by electro-elution from agarose gel: pWE.pac digested withPacI, pBr/AflII-Bam digested with PacI and BamHI and pBr/Ad.Bam-rITR#2digested with BamHI and PacI. These fragments were ligated together andpackaged using 1 phage packaging extracts (Stratagene) according to themanufacturer's protocol. After infection into host bacteria, colonieswere grown on plates and analyzed for presence of the complete insert.pWE/Ad.AflII-rITR contains all adenovirus type 5 sequences from bp 3534(AflII site) up to and including the right ITR (missing the most 3′ Gresidue).

[0046] pBr/Ad.1ITR-Sal (9.4) (ECACC deposit P97082115)

[0047] Adenovirus 5 wt DNA was treated with Klenow enzyme in thepresence of excess dNTPs and subsequently digested with SalI. Two of theresulting fragments, designated left ITR-Sal (9.4) and Sal (16.7)-rightITR, respectively, were isolated in LMP agarose (Seaplaque GTG), pBr322DNA was digested with EcoRV and SalI and treated with phosphatase (LifeTechnologies). The vector fragment was isolated using the Genecleanmethod (BIO 101, Inc) and ligated to the Ad5 SalI fragments. Only theligation with the 9.4 kb fragment gave colonies with an insert. Afteranalysis and sequencing of the cloning border a clone was chosen thatcontained the full ITR sequence and extended to the SalI site at bp9462.

[0048] pBr/Ad.1ITR-Sal (16.7) (ECACC deposit P97082118)

[0049] pBr/Ad.1ITR-Sal (9.4) is digested with SalI and dephosphorylated(TSAP, Life Technologies). To extend this clone up to the third SalIsite in Ad5, pBr/Ad.Cla-Bam was linearized with BamHI and partiallydigested with SalI. A 7.3 kb SalI fragment containing adenovirussequences from 9462-16746 was isolated in LMP agarose gel arid ligatedto the SalI-digested pBr/Ad. 1ITR-Sal (9.4) vector fragment.

[0050] pWE/Ad.AflII-EcoRI

[0051] pWE.pac was digested with ClaI and 5′ protruding ends were filledusing Klenow enzyme. The DNA was then digested with PacI and isolatedfrom agarose gel. pWE/AflII-rITR was digested with EcoRI and aftertreatment with Klenow enzyme digested with PacI. The large 24 kbfragment containing the adenoviral sequences was isolated from agarosegel and ligated to the ClaI-digested and blunted pWE.pac vector usingthe Ligation Express™ kit (Clontech). After transformation ofUltracompetent XL10-Gold cells from Stratagene, clones were identifiedthat contained the expected insert. pWE/AflII-EcoRI contains Ad5sequences from bp 3534-27336.

[0052] Construction of New Adapter Plasmids

[0053] The absence of sequence overlap between the recombinantadenovirus and E1 sequences in the packaging cell line is essential forsafe, RCA-free generation and propagation of new recombinant viruses.The adapter plasmid pMLPI.TK is an example of an adapter plasmiddesigned for use according to the invention in combination with theimproved packaging cell lines of the invention. This plasmid was used asthe starting material to make a new vector in which nucleic acidmolecules comprising specific promoter and gene sequences can be easilyexchanged. First, a PCR fragment was generated from pZipΔMo+PyF101 (N⁻)template DNA (described in PCT International Publication No. WO96/35798) with the following primers: LTR-1: 5′-CTG TAC GTA CCA GTG CATGG CCT AGG CAT GGA AAA ATA CAT AAC TG-3′ (SEQ ID NO:4) and LTR-2:5′-GCG GAT CCT TCG AAC CAT GGT AAG CTT GGT ACC GCT AGC GTT AAC CGG GCGACT CAG TCA ATC G-3′ (SEQ ID NO:5). Pwo DNA polymerase (BoehringerMannheim) was used according to manufacturer's protocol with thefollowing temperature cycles: once 5′ at 95° C.; 3′ at 55° C.; and 1′ at72° C., and 30 cycles of 1′ at 95° C., 1′ at 60° C., 1′ at 72° C.,followed by once 10′ at 72° C. The PCR product was then digested withBamHI and ligated into pMLP10 (Levrero et al. 1991) vector digested withPvuII and BamHI, thereby generating vector pLTR10. This vector containsadenoviral sequences from bp 1 up to bp 454 followed by a promoterconsisting of a part of the Mo-MuLV LTR having its wild-type enhancersequences replaced by the enhancer from a mutant polyoma virus (PyF101).The promoter fragment was designated L420. Next, the coding region ofthe murine HSA gene was inserted. pLTR10 was digested with BstBIfollowed by Klenow treatment and digestion with NcoI. The HSA gene wasobtained by PCR amplification on pUC18-HSA (Kay et al. 1990) using thefollowing primers:HSA1, 5′-GCG CCA CCA TGG GCA GAG CGA TGG TGG C-3′ (SEQID NO:6) and HSA2,5′-GTT AGA TCT AAG CTT GTC GAC ATC GAT CTA CTA ACA GTAGAG ATG TAG AA-3′ (SEQ ID NO:7). The 269 bp amplified fragment wassubcloned in a shuttle vector using the NcoI and BglII sites. Sequencingconfirmed incorporation of the correct coding sequence of the HSA gene,but with an extra TAG insertion directly following the TAG stop codon.The coding region of the HSA gene, including the TAG duplication wasthen excised as a NcoI (sticky)-SalI (blunt) fragment and cloned intothe 3.5 kb NcoI (sticky)/BstBI (blunt) fragment from pLTR10, resultingin pLTR-HSA10.

[0054] Finally, pLTR-HSA10 was digested with EcoRI and BamHI after whichthe fragment containing the left ITR, packaging signal, L420 promoterand HSA gene was inserted into vector pMLPI.TK digested with the sameenzymes and thereby replacing the promoter and gene sequences. Thisresulted in the new adapter plasmid pAd/L420-HSA that containsconvenient recognition sites for various restriction enzymes around thepromoter and gene sequences. SnaBI and AvrII can be combined with HpaI,NheI, KpnI, HindIII to exchange promoter sequences, while the lattersites can be combined with the ClaI or BamHI sites 3′ from HSA codingregion to replace genes in this construct.

[0055] Another adapter plasmid that was designed to allow easy exchangeof nucleic acid molecules was made by replacing the promoter, gene andpoly A sequences in pAd/L420-HSA with the CMV promoter, a multiplecloning site, an intron and a poly-A signal. For this purpose,pAd/L420-HSA was digested with AvrII and BglII followed by treatmentwith Klenow to obtain blunt ends. The 5.1 kb fragment with pBr322 vectorand adenoviral sequences was isolated and ligated to a blunt 1570 bpfragment from pcDNA1/amp (Invitrogen) obtained by digestion with HhaIand AvrII followed by treatment with T4 DNA polymerase. This adapterplasmid was named pCLIP.

[0056] Generation of Recombinant Adenoviruses

[0057] To generate E1 deleted recombinant adenoviruses with the newplasmid-based system, the following constructs were prepared: a) Anadapter construct containing the expression cassette with the nucleicacid of interest linearized with a restriction enzyme that cuts at the3′ side of the overlapping adenoviral genome fragment, preferably notcontaining any pBr322 vector sequences; and b) A complementingadenoviral genome construct pWE/Ad.AflII-rITR digested with PacI. Thesetwo DNA molecules were further purified by phenol/chloroform extractionand EtOH precipitation. Co-transfection of these plasmids into anadenovirus packaging cell line, generated recombinant replicationdeficient adenoviruses by a one-step homologous recombination betweenthe adapter and the complementing construct.

[0058] Alternatively, instead of pWE/Ad.AflII-rITR other fragments, canbe used, e.g., pBr/Ad.Cla-Bam digested with EcoRI and BamHI orpBr/Ad.AflII-BamHI digested with PacI and BamHI can be combined withpBr/Ad.Sal-rITR digested with SalI. In this case, three plasmids arecombined and two homologous recombinations are needed to obtain arecombinant adenovirus. It is to be understood that those skilled in theart may use other combinations of adapter and complementing plasmidswithout departing from the present invention. A general protocol asoutlined below and meant as a non-limiting example of the presentinvention has been performed to produce several recombinant adenovirusesusing various adapter plasmids and the Ad.AflII-rITR fragment.Adenovirus packaging cells (PER.C6) were seeded in ˜25 cm² flasks andthe next day when they were at ˜80% confluency, transfected with amixture of DNA and lipofectamine agent (Life Techn.) as described by themanufacturer. Routinely, 40 μl lipofectamine, 4 μg adapter plasmid and 4μg of the complementing adenovirus genome fragment AflII-rITR (or 2 μgof all three plasmids for the double homologous recombination) are used.Under these conditions transient transfection efficiencies of ˜50% (48hrs post transfection) are obtained as determined with controltransfections using a pAd/CMV-LacZ adapter. Two days later, cells arepassaged to ˜80 cm² flasks and further cultured. Approximately five (forthe single homologous recombination) to eleven days (for the doublehomologous recombination) later a cytopathogenic effect (CPE) is seen,indicating that functional adenovirus has formed. Cells and medium areharvested upon full CPE and recombinant virus is released byfreeze-thawing. An extra amplification step in an 80 cm² flask isroutinely performed to increase the yield since at the initial stage thetiters are found to be variable despite the occurrence of full CPE.After amplification, viruses are harvested and plaque purified on PER.C6cells. Individual plaques are tested for viruses with active transgenes.

[0059] Besides replacements in the E1 region it is possible to delete orreplace (part of) the E3 region in the adenovirus because E3 functionsare not necessary for the replication, packaging and infection of the(recombinant) virus. This creates the opportunity to use a larger insertor to insert more than one gene without exceeding the maximum packagesize (approximately 105% of wt genome length). This can be done, forexample, by deleting part of the E3 region in the pBr/Ad.Bam-rITR cloneby digestion with XbaI and religation. This removes Ad5 wt sequences28592-30470 including all known E3 coding regions. Another example isthe precise replacement of the coding region of gp19K in the E3 regionwith a polylinker allowing insertion of new sequences. This, 1) leavesall other coding regions intact and 2) obviates the need for aheterologous promoter since the transgene is driven by the E3 promoterand pA sequences, leaving more space for coding sequences. To this end,the 2.7 kb EcoRI fragment from wt Ad5 containing the 5′ part of the E3region was cloned into the EcoRI site of pBluescript (KS⁻) (Stratagene).Next, the HindIII site in the polylinker was removed by digestion withEcoRV and HindI and subsequent religation. The resulting clonepBS.Eco-Eco/ad5DHIII was used to delete the gp19K coding region. Primers1 (5′-GGG TAT TAG GCC AA AGG CGC A-3′) (SEQ ID NO:8) and 2 (5′-GAT CCCATG GAA GCT TGG GTG GCG ACC CCA GCG-3′) (SEQ ID NO:9) were used toamplify a sequence from pBS.Eco-Eco/Ad5DHIII corresponding to sequences28511 to 28734 in wt Ad5 DNA. Primers 3 (5′-GAT CCC ATG GGG ATC CTT TACTAA GTT ACA AAG CTA-3′) (SEQ ID NO:10) and 4 (5′-GTC GCT GTA GTT GGA CTGG-3′) (SEQ ID NO:11) were used on the same DNA to amplify Ad5 sequencesfrom 29217 to 29476. The two resulting PCR fragments were ligatedtogether by virtue of the new introduced NcoI site and subsequentlydigested with XbaI and MunI. This fragment was then ligated into thepBS.Eco-Eco/ad5ΔHIII vector that was digested with XbaI (partially) andMunI generating pBS.Eco-Eco/ad5ΔHIII.Δgp19K. To allow insertion offoreign genes into the HindIII and bamHI site, an XbaI deletion was madein pBS.Eco-Eco/ad5ΔHIII.iAgp19K to remove the BamHI site in theBluescript polylinker. The resulting plasmidpBS.Eco-Eco/ad5ΔHIIIΔgp19KΔXbaI, contains unique HindIII and BamHI sitescorresponding to sequences 28733 (HindIII) and 29218 (BamHI) in Ad5.After introduction of a foreign gene into these sites, either thedeleted XbaI fragment is reintroduced, or the insert is recloned intopBS.Eco-Eco/ad5ΔHIII.Δgp19K using HindIII and for example MunI. Usingthis procedure, we have generated plasmids expressing HSV-TK, hIL-1a,rat IL-3, luciferase or LacZ. The unique SrfI and NotI sites in thepBS.Eco-Eco/ad5ΔHIII.Δgp19K plasmid (with or without inserted gene ofinterest) are used to transfer the region comprising the gene ofinterest into the corresponding region of pBr/Ad.Bam-rITR, yieldingconstruct pBr/Ad.Bam-rITRΔ.gp19K (with or without inserted gene ofinterest). This construct is used as described supra to producerecombinant adenoviruses. In the viral context, expression of insertedgenes is driven by the adenovirus E3 promoter.

[0060] Recombinant viruses that are both E1 and E3 deleted are generatedby a double homologous recombination procedure as described above forE1-replacement vectors using a plasmid-based system consisting of:

[0061] a) an adapter plasmid for E1 replacement according to theinvention, with or without insertion of a first gene of interest,

[0062] b) the pWE/Ad.AflII-EcoRI fragment, and

[0063] c) the pBr/Ad.Bam-rITRΔgp19K plasmid with or without insertion ofa second gene of interest.

[0064] In addition to manipulations in the E3 region, changes of (partsof) the E4 region can be accomplished easily in pBr/Ad.Bam-rITR.Generation and propagation of such a virus, however, in some casesdemands complementation in trans.

Example 2 Generation of Adenovirus Serotype 5 Based Viruses WithChimeric Fiber Proteins

[0065] The method described infra to generate recombinant adenovirusesby co-transfection of two, or more separate cloned adenovirus sequences.One of these cloned adenovirus sequences was modified such that theadenovirus serotype 5 fiber DNA was deleted and substituted for uniquerestriction sites thereby generating ‘template clones’ which allow forthe easy introduction of DNA sequences encoding for fiber proteinderived from other adenovirus serotypes.

[0066] Generation of Adenovirus Template Clones Lacking DNA Encoding forFiber

[0067] The fiber coding sequence of adenovirus serotype 5 is locatedbetween nucleotides 31042 and 32787. To remove the adenovirus serotype 5DNA encoding fiber we started with construct pBr/Ad.Bam-rITR. First anNdeI site was removed from this construct. For this purpose, pBr322plasmid DNA was digested with NdeI after which protruding ends werefilled using Klenow enzym. This pBr322 plasmid was then re-ligated,digested with NdeI and transformed into E. coli DH5α. The obtainedpBr/ANdeI plasmid was digested with ScaI and SalI and the resulting 3198bp vector fragment was ligated to the 15349 bp ScaI-SalI fragmentderived from pBr/Ad.BamrITR, resulting in plasmid pBr/Ad.Bam-rITRΔNdeIwhich hence contained a unique NdeI site. Next a PCR was performed witholigonucleotides NY-up: 5′-CGA CAT ATG TAG ATG CAT TAG TTT GTG TTA TGTTTC AAC GTG-3′ (SEQ ID NO:12) and NY-down: 5′-GGA GAC CAC TGC CAT GTT-3′(SEQ ID NO:13). During amplification, both an NdeI (bold face) and anNsiI restriction site (underlined) were introduced to facilitate cloningof the amplified fiber DNAs. Amplification consisted of 25 cycles ofeach 45 sec. at 94° C., 1 min. at 60° C., and 45 sec. at 72° C. The PCRreaction contained 25 pmol of oligonucleotides NY-up or NY-down, 2 mMdNTP, PCR buffer with 1.5 mM MgCl₂, and 1 unit of Elongase heat stablepolymerase (Gibco, The Netherlands). One-tenth of the PCR product wasrun on an agarose gel that demonstrated that the expected DNA fragmentof ±2200 bp was amplified. This PCR fragment was subsequently purifiedusing Geneclean kit system (Bio 101 Inc). Then, both the constructpBr/Ad.Bam-rITRΔNdeI as well as the PCR product were digested withrestriction enzymes NdeI and SbfI. The PCR fragment was subsequentlycloned using T4 ligase enzyme into the NdeI and SbfI digestedpBr/Ad.Bam-rITRΔNdeI, generating pBr/Ad.BamRΔFib. This plasmid allowsinsertion of any PCR amplified fiber sequence through the unique NdeIand NsiI sites that are inserted in place of the removed fiber sequence.Viruses can be generated by a double homologous recombination inpackaging cells described infra using an adapter plasmid, constructpBr/Ad.AflII-EcoRI digested with PacI and EcoRI and a pBr/Ad.BamRΔFibconstruct in which heterologous fiber sequences have been inserted. Toincrease the efficiency of virus generation, the constructpBr/Ad.BamRΔFib was modified to generate a PacI site flanking the rightITR. Hereto, pBr/Ad.BamRΔFib was digested with AvrII and the 5 kbadenofragment was isolated and introduced into the vectorpBr/Ad.Bam-rITR.pac#8 replacing the corresponding AvrII fragment. Theresulting construct was named pBr/Ad.BamRΔFib.pac. Once a heterologousfiber sequence is introduced in pBr/Ad.BamRΔFib.pac, the fiber modifiedright hand adenovirus clone may be introduced into a large cosmid cloneas described for pWE/Ad.AflII-rITR in example 1. Such a large cosmidclone allows generation of adenovirus by only one homologousrecombination making the process extremely efficient.

[0068] Amplification of Fiber Sequences From Adenovirus Serotypes

[0069] To enable amplification of the DNAs encoding fiber proteinderived from alternative serotypes degenerate oligonucleotides weresynthesized. For this purpose, first known DNA sequences encoding forfiber protein of alternative serotypes were aligned to identifyconserved regions in both the tail-region as well as the knob-region ofthe fiber protein. From the alignment, which contained the nucleotidesequence of 19 different serotypes representing all 6 subgroups,(degenerate) oligonucleotides were synthesized (see Table II). Alsoshown in Table II is the combination of oligonucleotides used to amplifythe DNA encoding fiber protein of a specific serotype. The amplificationreaction (50 μl) contained 2 mM dNTPs, 25 pmol of each oligonucleotide,standard 1× PCR buffer, 1, 5 mM MgCl₂, and 1 Unit Pwo heat stablepolymerase (Boehringer) per reaction. The cycler program contained 20cycles, each consisting of 30 sec. 94° C., 60 sec. 60-64° C., and 120sec. At 72° C., 10% of the PCR product was run on an agarose gel whichdemonstrated that a DNA fragment was amplified. Of each differenttemplate, two independent PCR reactions were performed after which theindependent PCR fragments obtained were sequenced to determine thenucleotide sequence. From 11 different serotypes, the nucleotidesequence could be compared to sequences present in GenBank. Of all otherserotypes, the DNA encoding fiber protein was previously unknown and wastherefore aligned with known sequences from other subgroup members todetermine homology i.e. sequence divergence. Of the 51 human serotypesknown to date, all fiber sequences, except for serotypes 1, 6, 18, and26, have been amplified and sequenced.

[0070] Generation of Fiber Chimeric Adenoviral DNA Constructs

[0071] All amplified fiber DNAs as well as the vector (pBr/Ad.BamRΔFib)were digested with NdeI and NsiI. The digested DNAs were subsequentlyrun on a agarose gel after which the fragments were isolated from thegel and purified using the Geneclean kit (Bio 101 Inc). The PCRfragments were then cloned into the NdeI and NsiI sites ofpBr/AdBamRΔFib, thus generating pBr/AdBamRFibXX (where XX stands for theserotype number of which the fiber DNA was isolated). So far the fibersequence of serotypes5/7/8/9/10/11/12/13/14/16/17/19/21/24/27/28/29/30/32/33/34/35/36/37/38/40-s/40-L/41-S/42/45/47/49/51have been cloned into pBr/AdBamRFibXX. From pBr/AdBamRFibXX (where XX is5/8/9/10/11/13/16/17/24/27/30/32/33/34/35/38/40-S/40-L/45/47/49/51) acosmid clone in pWE/Ad.AflII-rITR (see example 1) was generated tofacilitate efficient virus generation. This cosmid cloning resulted inthe formation of construct pWE/Ad.AflII-rITR/FibXX (where XX stands forthe serotype number of which the fiber DNA was isolated).

[0072] Generation of Recombinant Adenovirus Chimeric for Fiber Protein

[0073] To generate recombinant adenovirus carrying the fiber of serotype12,16,28,40-L, 51, and 5, three constructs, pCLIP/luciferase,pWE/AdAflII-Eco and pBr/AdBamrITR.pac/fibXX (XX=12, 16, 28, 40-L, 51,and 5) were transfected into adenovirus producer cells. To generaterecombinant Ad 5 virus carrying the fiber of5/7/8/9/10/11/12/13/14/16/17/19/21/24/27/28/29/30/32/33/34/35/36/37/38/40-S/40-L/41-S/42/45/47/49/51,two constructs, pCLIP/luciferase and pWE/Ad.AflII-rITR/FibXX weretransfected into adenovirus producer cells. For transfection, 2 μg ofpCLIP/luciferase, and 4 μg of both pWE/AdAflII-Eco andpBr/AdBamrITR.pac/fibXX (or in case of cosmids; 4 μg of pCLIP/luciferaseplus 4 μg of pWE/Ad.AflII-rITR/FibXX) were diluted in serum free DMEM to100 μl total volume. To this DNA suspension 100 μl 1× dilutedLipofectamine (Gibco) was added. After 30 min at room temperature theDNA-lipofectamine complex solution was added to 2.5 ml of serum-freeDMEM which was subsequently added to a T25 cm² tissue culture flask.This flask contained 2×10⁶ PER.C6 cells that were seeded 24-hours priorto transfection. Two hours later, the DNA-lipofectamine complexcontaining medium was diluted once by the addition of 2.5 ml DMEMsupplemented with 20% fetal calf serum. Again 24 hours later the mediumwas replaced by fresh DMEM supplemented with 10% fetal calf serum. Cellswere cultured for 6-8 days, subsequently harvested, and freeze/thawed 3times. Cellular debris was removed by centrifugation for 5 min at 3000rpm room temperature. Of the supernatant (12.5 ml) 3-5 ml was used toinfect again infect PER.C6 cells (T80 cm² tissue culture flasks). Thisre-infection results in full cytopathogenic effect (CPE) after 5-6 daysafter which the adenovirus is harvested as described above. Besides theconstruction and generation of fiber-chimeric vectors carrying onlyluciferase as a marker gene as described above, many fiber-chimericviruses were generated carrying other marker genes by using pCLIP orpAdapt as adapter plasmids in which for instance LacZ or greenfluorescent protein (GFP) were cloned in the polylinker present in theseplasmids. Here, pCLIP/LacZ and pAdapt.GFP were used to generaterecombinant adenoviruses expressing the respective transgenes (thesedifferent adapter plasmids are described above and in PCT InternationalPublication Nos. WO 99/55132, WO 00/63403, and WO 01/20014).

Example 3 Production, Purification, and Titration of Fiber ChimericAdenoviruses

[0074] Of the supernatant obtained from transfected PER.C6 cells 10 mlwas used to inoculate a 1 liter fermentor which contained 1-1.5×10⁶cells/ml PER.C6 that were specifically adapted to grow in suspension.Three days after inoculation, the cells were harvested and pelleted bycentrifugation for 10 min. at 1750 rpm at room temperature. The chimericadenoviruses present in the pelleted cells were subsequently extractedand purified using the following downstream processing protocol. Forsmall scale productions adherent PER.C6 cells were used in combinationwith T175 cm² tissue culture flasks. Irrespective of the scale of theproduction cells were treated identically. The pellet was dissolved in50 ml 10 mM NaPO₄ and frozen at −20° C. After thawing at 37° C., 5.6 mldeoxycholate (5% w/v) was added after which the solution washomogenized. The solution was subsequently incubated for 15 min. at 37°C. to completely crack the cells. After homogenizing the solution, 1875μl (1M) MgCl₂ was added and 5 ml 100% glycerol. After the addition of375 μl DNAse (10 mg/ml) the solution was incubated for 30 min. at 37° C.Cell debris was removed by centrifugation at 1880 g for 30 min. at roomtemperature without the brake on. The supernatant was subsequentlypurified from proteins by loading on 10 ml of Freon™. Uponcentrifugation for 15 min. at 2000 rpm without brake at room temperaturethree bands are visible of which the upper band represents theadenovirus. This band was isolated by pipetting after which it wasloaded on a Tris/HC1 (1M) buffered cesium chloride block gradient(range: 1.2 to 1.4 g/ml). Upon centrifugation at 21,000 rpm for 2.5 h at10° C. the virus was purified from remaining protein and cell debrissince the virus, in contrast to the other components, does not migrateinto the 1.4 g/ml cesium chloride solution. The virus band is isolatedafter which a second purification using a Tris/HC1 (1M) bufferedcontinues gradient of 1.33 g/ml of cesium chloride is performed. Aftervirus loading on top of this gradient the virus is centrifuged for 17 hat 55000 rpm at 10° C. Subsequently the virus band is isolated and afterthe addition of 30 μl of sucrose (50 w/v) excess cesium chloride isremoved by three rounds of dialysis, each round comprising of 1 h. Fordialysis the virus is transferred to dialysis slides (Slide-a-lizer, cutoff 10,000 kDa, Pierce, USA). The buffers used for dialysis are PBSwhich are supplemented with an increasing concentration of sucrose(round 1 to 3: 30 ml, 60 ml, and 150 ml sucrose (50% w/v)/1.5 liter PBS,all supplemented with 7.5 ml 2% (w/v) CaMgCl₂). After dialysis, thevirus is removed from the slide-a-lizer after which it is aliquoted inportions of 25 and 100 μupon which the virus is stored at −85° C. Todetermine the number of virus particles per ml, 100 μl of the virusbatch is run on a high pressure liquid chromatograph (HPLC). Theadenovirus is bound to the column (anion exchange) after which it iseluted using a NaCl gradient (range 300-600 mM). Thy determining thearea under the virus peak the number of virus particles can becalculated. To determine the number of infectious units (1 U) per mlpresent in a virus batch, titrations are performed on 911 cells. Forthis purpose, 4×10⁴ 911 cells are seeded per well of 96-well plates inrows B, D, and F in a total volume of 100 μl.per well. Three hours afterseeding the cells are attached to the plastic support after which themedium can be removed. A volume of 200 μl is added to the cells, induplicate, containing different dilutions of virus (range: 10² timesdiluted to 2×10⁹). By screening for CPE, the highest virus dilution thatstill renders CPE after 14 days is considered to contain at least oneinfectious unit. Using this observation, together with the calculatedamount of virus volume present in these wells renders the number ofinfectious units per ml of a given virus batch. The production results,i.e., virus particles per ml of chimeric adenoviruses with theluciferase cDNA as a marker, are shown in Table II.

Example 4 Expression of Integrins and CAR On Human Primary Chondrocytes

[0075] To test for expression on primary chondrocytes for membranemolecules known to be involved in Ad5 infection, the presence of CAR,and a_(v)-integrins was assayed on a flow cytometer. Since the MRC classI alpha-2 domain has also been proposed as a receptor for Ad5,expression of this molecule was tested as well. For this purpose 2×10⁴chondrocytes were washed once with PBS/0.5% BSA after which the cellswere centrifuged for 5 min. at 1750 rpm at room temperature.

[0076] Subsequently, 10 μl of a 100 times diluted a_(v)b3 antibody (Mab1961, Brunswick Chemie, NL), a 100 times diluted antibody a_(v)b5(antibody (Mab 1976, Brunswick Chemie, NL), or 1000 times diluted CAR(Hsu et al. 1988) antibody (a kind gift of Dr. Bergelson, HarvardMedical School, USA) was added to the cell pellet after which the cellswere incubated for 30 min. at 4° C. in a dark environment. After thisincubation, cells were washed twice with PBS/0.5% BSA and again pelletedby centrifugation for 5 min. at 1750 rpm room temperature. To label thecells, 10 ml of rat antimouse IgG1 labeled with phycoerythrine (PE) wasadded to the cell pellet upon which the cells were again incubated for30 min. at 4° C. in a dark environment. Finally the cells were washedtwice with PBS/0.5% BSA and analyzed on a flow cytometer. The results ofthese experiments are shown in FIG. 1. From the results it can beconcluded that primary human chondrocytes do not express detectablelevels of CAR that is the primary receptor for Ad5. The cells do expressMHC-class I, but since this is a very low affinity receptor, the resultsconfirm that chondrocytes are difficult to transduce with an adenovirusserotype 5.

Example 5 Adenovirus Transduction of Human Primary Chondrocytes

[0077] Human primary chondrocytes were cultured in Dulbecco's modifiedEagles medium (DMEM) supplemented with 10% fetal calf serum and furthersupplemented with essential amino acids (proline 0.4 mM), non-essentialamino acids (1×), cholic acid-6-phosphate (0.2 mM) and buffered withHEPES (10 nM) (all materials derived from Gibco). In a first experiment,10⁵ chondrocytes were seeded in wells of 24-well plates. The next daycells were exposed to either 100, 500, or 1000 virus particles per cellof recombinant fiber chimeric viruses carrying the fiber of serotype 8,9, 10, 11, 12, 13, 16, 17, 24, 27, 30, 32, 33, 35, 38, 40-S, 40-L, 45,47, 49, or 51. In these experiments, the parent vector (fib5) was takenalong as a reference. Forty-eight hours after the addition of virus,cells were washed twice with 1 ml PBS after which cells were lysed byadding 100 μl of cell lysis buffer. Lysates were subsequentlytransferred to 96-well plates and stored at −20 degrees Celsius untilluciferase activity measurement. Luciferase activity was determinedusing a bioluminescence machine, the luciferase assay kit from Promega™(catalog no. E-1501) and the instructions provided by the manufacturer.The results of the luciferase transgene expression measured in primaryhuman chondrocytes after transduction with the panel of fiber chimericviruses is shown in FIGS. 2A and 2B (chondrocytes derived from twoindividual donors). The results demonstrate that several fiber chimericviruses perform better on chondrocytes as compared to the parent vector(Ad5). These viruses carry the fiber from a subgroup B viruses i.e. 11,16,35, and 51. Also, several, but not all, viruses carrying a fiberoriginating from subgroup D, i.e., 8, 13, and 32 are better equipped fortransducing chondrocytes. These results thus clearly show that from alibrary containing different fiber-chimeric vectors, adenoviruses can beidentified that are improved in their ability to transduce human celltypes of interest, i.e. chondrocytes. Moreover, these resultsdemonstrate that human adenoviruses, between subgroups but even within asubgroup, can recognize distinct attachment molecules on human cells.

Example 6 Effect of Adenovirus Transduction On Human Chondrocytes

[0078] To determine the effect of infection by adenovirus in time onchondrocytes in terms of toxicity, human primary chondrocytes wereseeded 24 h prior to infection in a density of 5×10⁴ cells/well in a 24well dish. Cells were infected with one of the improved virusesidentified, i.e. Ad5Fib16. Chondrocytes were infected with Ad5Fib16LacZusing moi's of 100, 1000 and 5000 vp/cell. The viruses remained in thesolution throughout the experiment. LacZ expression was monitored atdifferent time points: 20, 44, 68, 140, 164 and 188 post infection. Theviability of the cells was determined by using the MTS assay fromPromega using the instructions provided by the manufacturer. In general,200 μl of the so-called Cell Titer Aqueous One Solution Reagent wasadded to the well and incubated for 4 h at 37° C. This was followed byinactivation of the virus and stopping the reaction by adding 25 μl 10%SDS solution. 100 μl of the mixture was transferred to a well in a96-well dish and absorbance was measured at 490 nm and compared to thecontrols. Every time point experiment was performed in triplicate andaveraged. The results are shown in FIG. 3A and indicate that the humanprimary chondrocytes do not suffer significantly from the adenoviralinfections, even when an high moi of 5000 virus particles per cell wasapplied.

[0079] Cells that were infected in an identical manner as describedabove, were used to determine the expression of the LacZ transgene overtime. For this, cells were washed twice with PBS and fixed with 0.5ml/well of a fixative solution (1.08 ml formaldehyde (JT Baker) plus 480μl Glutar-dialdehyde (Merck) in 40 ml PBS) and incubated for 10 min. atroom temperature. Then, cells were washed twice with PBS and stainedwith 0.5 ml/well staining solution (1 ml K₃Fe(CN)₆, 1 ml K₄Fe(CN)₆, 80μl 1M MgCl₂, filled up to 40 ml with PBS, to which 150 μl X-Gal per 6 mlis added prior to use) for 4 h at 37° C. Positive cells were counted andcompared to negative cells. The results are shown in FIG. 3B andindicate that over prolonged periods of time the transgene expression isnot significantly diminished in human primary chondrocytes uponinfection with adenovirus. The latter results indicate that sufficientvector copies are present in the nucleus of transduced chondrocytes toallow for at least 4 cell doublings without losing the marker gene.

Example 7 Expression of Green Fluorescent Protein In Human ChondrocytesUpon Infection By Adenovirus.

[0080] Although both luciferase and LacZ provide data concerning thetransduction efficiency of Ad5 and fiber-chimeric vectors inchondrocytes determination on a single cell level is difficult usingthese marker genes. Therefore, we compared Ad5 with Ad5Fib13, Ad5Fib16,Ad5Fib35, and Ad5Fib51 viruses all carrying green fluorescent protein(GFP) as a marker gene. Detection of GFP expression can be monitoredusing a flow cytometer (FACScalibur, Becton Dickinson). Hereto, humanprimary chondrocytes were seeded 24 h prior to infection in a density of5×10⁴ cells/well in a 24 well dish. Cells were exposed for 2 h to thevectors at a concentration 100, 500, 1000, virus particles per cell.Forty-eight hours after virus exposure cells were harvested andsubjected to flow cytometric analysis. To set the flow cytometer,non-transduced chondrocytes were used (background gate 1% positivecells). Subsequently, cells exposed to the different adenoviral vectorswere assayed. As shown in FIG. 4A, the percentage of cells positive forGFP increased using an increase in viral load from 100 to 1000 virusparticles per cell. Moreover, the results show that using Ad5Fib16,Ad5Fib35, or Ad5Fib51 the amount of cells transduced is increaseddrastically as compared to cells exposed to Ad5. (3-9 fold more cellsdependent on MOI). Besides the increase in the number of cells thatbecame positive for GFP using the fiber-chimeric vectors anotherparameter was also monitored, i.e. median fluorescence. This parametergives information concerning the amount of GFP produced on a single celllevel. Results of this analysis is shown in FIG. 4B as demonstrates thatthe amount of GFP produced per cell is much higher using Ad5Fib16,Ad5Fib35, or Ad5Fib51 as compared to Ad5. Thus, using thesefiber-chimeric vectors both the amount of cells transduced as well asthe amount of vector copies per cell is significantly increased.

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[0123] TABLE II Production results of recombinant fiber chimericadenoviruses. Results in virus particles per milliliter as determined byHPLC. Adenovirus Virus particles/ml Ad5Fib5 2.2 × 10¹² Ad5Fib9 4.9 ×10¹¹ Ad5Fib10 5.5 × 10¹¹ Ad5Fib11 1.1 × 10¹² Ad5Fib12 4.4 × 10¹²Ad5Fib13 1.1 × 10¹² Ad5Fib16 1.4 × 10¹² Ad5Fib17 9.3 × 10¹¹ Ad5Fib24 1.0× 10¹² Ad5Fib27 3.0 × 10¹¹ Ad5Fib30 7.1 × 10¹¹ Ad5Fib32 2.0 × 10¹²Ad5Fib33 1.5 × 10¹² Ad5Fib35 2.0 × 10¹² Ad5Fib38 5.8 × 10¹¹ Ad5Fib40-S3.2 × 10¹⁰ Ad5Fib40-L 2.0 × 10¹² Ad5Fib45 2.8 × 10¹² Ad5Fib47 2.6 × 10¹²Ad5Fib49 1.2 × 10¹² Ad5Fib51 5.1 × 10¹²

What is claimed is:
 1. A method of delivering a nucleic acid of interestto a chondrocyte, said method comprising: providing a recombinantadenovirus having a tropism for chondrocytes, said recombinantadenovirus comprising: a nucleic acid of interest; and a nucleic acidencoding at least a part of a fiber protein of a B-type adenovirus,wherein said fiber protein of the B-type adenovirus has a tropism forchondrocytes; and infecting a chondrocyte with said recombinantadenovirus, such that said nucleic acid is delivered to saidchondrocyte.
 2. The method according to claim 1, wherein said B-typeadenovirus is selected from the group consisting of adenovirus type 16,adenovirus type 35, and adenovirus type
 51. 3. The method according toclaim 1, wherein said recombinant adenovirus comprises an adenovirustype 5 nucleic acid sequence.
 4. The method according to claim 3,wherein said recombinant adenovirus comprises an adenovirus type 5genome.
 5. The method according to claim 1, wherein said recombinantadenovirus comprises at least one deletion in the E1 or the E3 region,where the nucleic acid of interest is inserted or can be inserted. 6.The method according to claim 1, wherein said nucleic acid of interestencodes at least one amino acid sequence that inhibits cartilage diseaseprogression and/or at least one amino acid sequence that counteracts theloss of cartilage.
 7. The method according to claim 1, wherein thenucleic acid of interest encodes at least one member of the family ofbone morphogenesis proteins.
 8. A chondrocyte provided with anadditional nucleic acid of interest encoding at least one amino acidsequence that inhibits cartilage disease progression and/or at least oneamino acid sequence that counteracts the loss of cartilage, saidadditional nucleic acid of interest being provided by the methodaccording to claim
 6. 9. A chondrocyte provided with an additionalnucleic acid of interest encoding at least one member of the family ofbone morphogenesis proteins, said additional nucleic acid of interestbeing provided by the method according to claim
 7. 10. A method ofinhibiting cartilage disease progression in a subject, said methodcomprising: preparing a recombinant adenovirus having a tropism forchondrocytes, said recombinant adenovirus comprising: a nucleic acid ofinterest encoding at least one amino acid sequence that inhibitscartilage disease progression and/or at least one amino acid sequencethat counteracts cartilage loss; and a nucleic acid encoding at least apart of a fiber protein of a B-type adenovirus, wherein said fiberprotein of the B-type adenovirus has a tropism for chondrocytes; andinfecting a chondrocyte with said recombinant adenovirus, such that saidnucleic acid of interest is expressed in said chondrocyte, therebyinhibiting cartilage disease progression.
 11. The method according toclaim 10, wherein said B-type adenovirus is selected from the groupconsisting of adenovirus type 16, adenovirus type 35, and adenovirustype
 51. 12. The method according to claim 10, wherein said recombinantadenovirus comprises an adenovirus type 5 nucleic acid sequence.
 13. Themethod according to claim 12, wherein said recombinant adenoviruscomprises an adenovirus type 5 genome.
 14. The method according to claim11, wherein said recombinant adenovirus comprises at least one deletionin the E1 or the E3 region, where the nucleic acid of interest isinserted or can be inserted.
 15. A method of repairing cartilage, saidmethod comprising the steps of: preparing a recombinant adenovirushaving a tropism for chondrocytes, said recombinant adenoviruscomprising: a nucleic acid of interest encoding at least one member ofthe family of bone morphogenesis proteins; and a nucleic acid encodingat least a part of a fiber protein of a B-type adenovirus, wherein saidfiber protein of the B-type adenovirus has a tropism for chondrocytes;and infecting a chondrocyte with said recombinant adenovirus, such thatsaid nucleic acid of interest is expressed in said chondrocyte, therebyeffecting cartilage repair.
 16. The method according to claim 15,wherein said B-type adenovirus is selected from the group consisting ofadenovirus type 16, adenovirus type 35, and adenovirus type
 51. 17. Themethod according to claim 15, wherein said recombinant adenoviruscomprises an adenovirus type 5 nucleic acid sequence.
 18. The methodaccording to claim 17, wherein said recombinant adenovirus comprises anadenovirus type 5 genome.
 19. The method according to claim 15, whereinsaid recombinant adenovirus comprises at least one deletion in the E1 orthe E3 region, where the nucleic acid of interest is inserted or can beinserted.